All adult tissues contain stem cells. Some tissues, like bone marrow and skin, harbor more adult stem cells; other tissues, like muscle, have fewer. When a tissue or organ is injured these stem cells possess a remarkable ability to divide and multiply. In the end, the ability of a tissue to repair itself seems to depend on how many stem cells reside in a particular tissue, and the state of those stem cells. For example, stress, disease, and aging all diminish the capacity of adult stem cells to self-renew and to proliferate, which in turn hinders tissue regeneration.

Our strategy is to commandeer the molecular machinery responsible for adult stem cell self-renewal and proliferation and by doing so, stimulate the endogenous program of tissue regeneration. This approach takes advantage of the solution that Nature itself developed for repairing damaged or diseased tissues, and controls adult stem cell proliferation in a localized, highly controlled fashion. This strategy circumvents the immunological, medical, and ethical hurdles that exist when exogenous stem cells are introduced into a human. When utilizing this strategy the goal of reaching clinical trials in human patients within 5 years becomes realistic.

Specifically, we will target the growing problem of neurologic, musculoskeletal, cardiovascular, and wound healing diseases by local delivery of a protein that promotes the body’s inherent ability to repair and regenerate tissues. We have evidence that this class of proteins, when delivered locally to an injury site, is able to stimulate adult tissue stem cells to grow and repair/replace the deficient tissue following injury. We have developed technologies to package the protein in a specialized manner that preserves its biological activity but simultaneously restricts its diffusion to unintended regions of the body. For example, when we treat a skeletal injury with this packaged protein we augment the natural ability to heal bone by 350%; and when this protein is delivered to the heart immediately after an infarction cardiac output is improved and complications related to scarring are reduced. This remarkable capacity to augment tissue healing is not limited to bones and the heart: the same powerful effect can be elicited in the brain, and skin injuries.

The disease targets of stroke, bone fractures, heart attacks, and skin wounds and ulcers represent an enormous health care burden now, but this burden is expected to skyrocket because our population is quickly aging. Thus, our proposal addresses a present and ongoing challenge to healthcare for the majority of Californians, with a novel therapeutic strategy that mimics the body’s inherent repair mechanisms.

Statement of Benefit to California:

Californians represent 1 in 7 Americans, and make up the single largest healthcare market in the United States. The diseases and injuries that affect Californians affect the rest of the US, and the world. For example, stroke is the third leading cause of death, with more than 700,000 people affected every year. It is a leading cause of serious long-term disability, with an estimated 5.4 million stroke survivors currently alive today. Symptoms of musculoskeletal disease are the number two most cited reasons for visit to a physician. Musculoskeletal disease is the leading cause of work-related and physical disability in the United States, with arthritis being the leading chronic condition reported by the elderly. In adults over the age of 70, 40% suffer from osteoarthritis of the knee and of these nearly 80% have limitation of movement. By 2030, nearly 67 million US adults will be diagnosed with arthritis. Cardiovascular disease is the leading cause of death, and is a major cause of disability worldwide. The annual socioeconomic burden posed by cardiovascular disease is estimated to exceed $400 billion annually and remains a major cause of health disparities and rising health care costs. Skin wounds from burns, trauma, or surgery, and chronic wounds associated with diabetes or pressure ulcer, exact a staggering toll on our healthcare system: Burns alone affect 1.25M Americans each year, and the economic global burden of these injuries approaches $50B/yr. In California alone, the annual healthcare expenditures for stroke, skeletal repair, heart attacks, and skin wound healing are staggering and exceed 700,000 cases, 3.5M hospital days, and $34B.

We have developed a novel, protein-based therapeutic platform to accelerate and enhance tissue regeneration through activation of adult stem cells. This technology takes advantage of a powerful stem cell factor that is essential for the development and repair of most of the body’s tissues. We have generated the first stable, biologically active recombinant Wnt pathway agonist, and showed that this protein has the ability to activate adult stem cells after tissue injury. Thus, our developmental candidate leverages the body’s natural response to injury. We have generated exciting preclinical results in a variety of animals models including stroke, skeletal repair, heart attack, and skin wounding. If successful, this early translational award would have enormous benefits for the citizens of California and beyond.

Progress Report:

In the first year of CIRM funding our objectives were to optimize the activity of the Wnt protein for use in the body and then to test, in a variety of injury models, the effects of this lipid-packaged form of Wnt. We have made considerable progress on both of these fronts. For example, in Roel Nusse and Jill Helms’ groups, we have been able to generate large amounts of the mouse form of Wnt3a protein and package it into liposomal vesicles, which can then be used by all investigators in their studies of injury and repair. Also, Roel Nusse succeeded in generating human Wnt3a protein. This is a major accomplishment since our ultimate goal is to develop this regenerative medicine tool for use in humans. In Jill Helms’ lab we made steady progress in standardizing the activity of the liposomal Wnt3a formulation, and this is critically important for all subsequent studies that will compare the efficacy of this treatment across multiple injury repair scenarios.
Each group began testing the effects of liposomal Wnt3a treatment for their particular application. For example, in Theo Palmer’s group, the investigators tested how liposomal Wnt3a affected cells in the brain following a stroke. We previously found that Wnt3A promotes the growth of neural stem cells in a petri dish and we are now trying to determine if delivery of Wnt3A can enhance the activity of endogenous stem cells in the brain and improve the level of recovery following stroke. Research in the first year examined toxicity of a liposome formulation used to deliver Wnt3a and we found it to be well tolerated after injection into the brains of mice. We also find that liposomal Wnt3a can promote the production of new neurons following stroke. The ongoing research involves experiments to determine if these changes in stem cell activity are accompanied by improved neurological function. In Jill Helms’ group, the investigators tested how liposomal Wnt3a affected cells in a bone injury site. We made a significant discovery this year, by demonstrating that liposomal Wnt3a stimulates the proliferation of skeletal progenitor cells and accelerates their differentiation into osteoblasts (published in Science Translational Medicine 2010). We also started testing liposomal Wnt3a for safety and toxicity issues, both of which are important prerequisites for use of liposomal Wnt3a in humans. Following a heart attack (i.e., myocardial infarction) we found that endogenous Wnt signaling peaks between post-infarct day 5-7. We also found that small aggregates of cardiac cells called cardiospheres respond to Wnt in a dose-responsive manner. In skin wounds, we tested the effect of boosting Wnt signaling during skin wound healing. We found that the injection of Wnt liposomes into wounds enhanced the regeneration of hair follicles, which would otherwise not regenerate and make a scar instead. The speed and strength of wound closure are now being measured.
In aggregate, our work on this project continues to move forward with a number of great successes, and encouraging data to support our hypothesis that augmenting Wnt signaling following tissue injury will provide beneficial effects.

In the second year of CIRM funding our objectives were to optimize packaging of the developmental candidate, Wnt3a protein, and then to continue to test its efficacy to enhance tissue healing. We continue to make considerable progress on the stated objectives. In Roel Nusse’s laboratory, human Wnt3a protein is now being produced using an FDA-approved cell line, and Jill Helms’ lab the protein is effectively packaged into lipid particles that delay degradation of the protein when it is introduced into the body.
Each group has continued to test the effects of liposomal Wnt3a treatment for their particular application. In Theo Palmer’s group we have studied how liposomal Wnt3a affects neurogenesis following stroke. We now know that liposomal Wnt3a transiently stimulates neural progenitor cell proliferation. We don’t see any functional improvement after stroke, though, which is our primary objective.
In Jill Helms’ group we’ve now shown that liposomal Wnt3a enhances fracture healing and osseointegration of dental and orthopedic implants and now we demonstrate that liposomal Wnt3a also can improve the bone-forming capacity of bone marrow grafts, especially when they are taken from aged animals.
We’ve also tested the ability of liposomal Wnt3a to improve heart function after a heart attack (i.e., myocardial infarction). Small aggregates of cardiac progenitor cells called cardiospheres proliferate to Wnt3a in a dose-responsive manner, and we see an initial improvement in cardiac function after treatment of cells with liposomal Wnt3a. the long-term improvements, however, are not significant and this remains our ultimate goal. In skin wounds, we tested the effect of boosting Wnt signaling during wound healing. We found that the injection of liposomal Wnt3a into wounds enhanced the regeneration of hair follicles, which would otherwise not regenerate and make a scar instead. The speed of wound closure is also enhanced in regions of the skin where there are hair follicles.
In aggregate, our work continues to move forward with a number of critical successes, and encouraging data to support our hypothesis that augmenting Wnt signaling following tissue injury will provide beneficial effects.

Every adult tissue harbors stem cells. Some tissues, like bone marrow and skin, have more adult stem cells and other tissues, like muscle or brain, have fewer. When a tissue is injured, these stem cells divide and multiply but only to a limited extent. In the end, the ability of a tissue to repair itself seems to depend on how many stem cells reside in a particular tissue, and the state of those stem cells. For example, stress, disease, and aging all diminish the capacity of adult stem cells to respond to injury, which in turn hinders tissue healing. One of the great unmet challenges for regenerative medicine is to devise ways to increase the numbers of these “endogenous” stem cells, and revive their ability to self-renew and proliferate.

The scientific basis for our work rests upon our demonstration that a naturally occurring stem cell growth factor, Wnt3a, can be packaged and delivered in such a way that it is robustly stimulates stem cells within an injured tissue to divide and self-renew. This, in turn, leads to unprecedented tissue healing in a wide array of bone injuries especially in aged animals. As California’s population ages, the cost to treat such skeletal injuries in the elderly will skyrocket. Thus, our work addresses a present and ongoing challenge to healthcare for the majority of Californians and the world, and we do it by mimicking the body’s natural response to injury and repair.

To our knowledge, there is no existing technology that displays such effectiveness, or that holds such potential for the stem cell-based treatment of skeletal injuries, as does a L-Wnt3a strategy. Because this approach directly activates the body’s own stem cells, it avoids many of the pitfalls associated with the introduction of foreign stem cells or virally reprogrammed autologous stem cells into the human body. In summary, our data show that L-Wnt3a constitutes a viable therapeutic approach for the treatment of skeletal injuries, especially those in individuals with diminished healing potential.

This progress report covers the period between Sep 01 2012through Aug 31 2013, and summarizes the work accomplished under ET funding TR1-01249. Under this award we developed a Wnt protein-based platform for activating a patient’s own stem cells for the purpose of tissue regeneration.

At the beginning of our grant period we generated research grade human WNT3A protein in quantities sufficient for all our discovery experiments. We then tested the ability of this WNT protein therapeutic to improve the healing response in animal models of stroke, heart attack, skin wounding, and bone fracture. These experimental models recapitulated some of the most prevalent and debilitating human diseases that collectively, affect millions of Californians.

At the end of year 2, we assembled an external review panel to select the promising clinical indication. The scientific advisory board unanimously selected skeletal repair as the leading indication. The WNT protein is notoriously difficult to purify; consequently in year 3 we developed new methods to streamline the purification of WNT proteins, and the packaging of the WNT protein into liposomal vesicles that stabilized the protein for in vivo use.

In years 3 and 4 we continued to accrue strong scientific evidence in both large and small animal models that a WNT protein therapeutic accelerates bone regeneration in critical size bony non-unions, in fractures, and in cases of implant osseointegration. In this last year of funding, we clarified and characterized the mechanism of action of the WNT protein, by showing that it activates endogenous stem cells, which in turn leads to faster healing of a range of different skeletal defects.

In this last year we also identified a therapeutic dose range for the WNT protein, and developed a route and method of delivery that was simultaneously effective and yet limited the body’s exposure to this potent stem cell factor. We initiated preliminary safety studies to identify potential risks, and compared the effects of WNT treatment with other commercially available bone growth factors. In sum, we succeeded in moving our early translational candidate from exploratory studies to validation, and are now ready to enter into the IND-enabling phase of therapeutic candidate development.

This progress report covers the period between Sep 01 2013 through April 30 2014, and summarizes the work accomplished under ET funding TR101249. Under this award we developed a Wnt protein-based platform for activating a patient’s own stem cells for purposes of tissue regeneration.

At the beginning of our grant period we generated research grade human WNT3A protein in quantities sufficient for all our discovery experiments. We then tested the ability of this WNT protein therapeutic to improve the healing response in animal models of stroke, heart attack, skin wounding, and bone fracture. These experimental models recapitulated some of the most prevalent and debilitating human diseases that collectively, affect millions of Californians. At the conclusion of Year 2 an external review panel was assembled and charged with the selection of a single lead indication for further development. The scientific advisory board unanimously selected skeletal repair as the lead indication.

In year 3 we accrued addition scientific evidence, using both large and small animal models, demonstrating that a WNT protein therapeutic accelerated bone healing. Also, we developed new methods to streamline the purification of WNT proteins, and improved our method of packaging of the WNT protein into liposomal vesicles (e.g., L-WNT3A) for in vivo use.

In year 4 we clarified the mechanism of action of L-WNT3A, by demonstrating that it activates endogenous stem cells and therefore leads to accelerated bone healing. We also continued our development studies, by identifying a therapeutic dose range for L-WNT3A, as well as a route and method of delivery that is both effective and safe. We initiated preliminary safety studies to identify potential risks, and compared the effects of L-WNT3A with other, commercially available bone growth factors.

In year 5 we initiated two new preclinical studies aimed at demonstrating the disease-modifying activity of L-WNT3A in spinal fusion and osteonecrosis. These two new indications were chosen by a CIRM review panel because they represent an unmet need in California and the nation. We also initiated development of a scalable manufacturing and formulation process for both the WNT3A protein and L-WNT3A formulation. These two milestones were emphasized by the CIRM review panel to represent major challenges to commercialization of L-WNT3A; consequently, accomplishment of these milestones is a critical yardstick by which progress towards an IND filing can be assessed.

With regards to objective 1, we employed established animal models of spinal fusion and osteonecrosis to demonstrate the disease-mitigating activity of our Developmental Candidate, autograftWNT.
With regards to objective 2, the mechanism of L-WNT3A action has been demonstrated in ex vivo, non-GLP pharmacology studies.
With regards to objective 3, we have completed characterization of the non-GLP substance, WNT3A drug product, and the drug product, L-WNT3A. Methods for reproducible and scaleable research grade production of the drug substance WNT3A, and the drug product, L-WNT3A, have been developed (see below for details). A serum free process has been achieved.